The PCR amplicon was sequenced in both directions by using the BigDye Terminator Cycle Sequencing kit (PerkinElmer-Applied Biosystems, Inc., Foster City, CA, USA) on an automated sequencer (ABI 3100; PerkinElmer-Applied Biosystems, Inc.)... The sequence of CMP034 was compared with those of reference strains available in the National Center for Biotechnology Information GenBank database by using BLAST (www.ncbi.nlm.nih.gov/blast)... The NSP4 nucleotide sequence of the CMP034 strain was deposited in GenBank under accession no... DQ534017... However, CMP034 and P21–5 shared a low degree of sequence identity with other NSP4 genetic groups... The phylogenetic tree confirmed that PoRV strains CMP034 and P21–5 were located exclusively in a separated branch, which was distantly related to the other 5 known NSP4 genetic groups (Figure)... The NSP4 sequence analysis of our CMP034 strain revealed a PoRV strain closely related genetically to the NSP4 gene sequence of PoRV strain P21–5 isolated in Slovenia... The only difference observed between the 2 strains was that CMP034 belonged to the G2-like genotype whereas P21–5 belonged to G1 genotype... The relatedness between NSP4 sequences of strains CMP034 and P21–5 was confirmed by phylogenetic analysis, which showed that both CMP034 and P21–5 clustered closely together in a branch separated from those of other 5 NSP4 genetic groups... This finding suggests that NSP4 of PoRV strain CMP034 and P21–5 may have derived from the same ancestor... The isolation of 2 strains of rotaviruses with a close genetic relatedness of NSP4 gene from Thailand and Slovenia, 2 countries that are located in different continents, may indicate that this novel NSP4 genetic group has already been introduced into PoRVs worldwide.

Figure 1: Phylogenetic analyses of the NSP4 nucleotide (A) and amino acid (B) sequences displaying the relationships between porcine rotavirus strain CMP034 (shown in boldface), P21–5, and other 5 known NSP4 genetic groups. Bootstrap values are shown at the branch nodes. Branch length for a 10% nucleotide difference is indicated at the bottom.

Mentions:
The complete NSP4 nucleotide sequence of PoRV CMP034 strain was 750 bp and contained a single long open reading frame coding for a protein of 175 aa. Comparative analysis of the CMP034 NSP4 sequence with those of the 5 representative established genetic groups (A–E) showed the highest sequence identity, at 92.6% nt and 96.9% aa levels, with 1 PoRV strain, P21–5 (9). However, CMP034 and P21–5 shared a low degree of sequence identity with other NSP4 genetic groups. The NSP4 sequence identities of the CMPO34 and P21-5 strains ranged from 74% to 78% nt and 75%–79% aa levels with those of genetic group A; 77%–86% nt and 79%–86% aa levels with genetic group B; 69%–73% nt and 75%–78% aa levels with genetic group C; 62%–65% nt and 55%–60% aa levels with genetic group D; and only 43%–50% nt and 29%–33% aa levels with genetic group E. The phylogenetic tree confirmed that PoRV strains CMP034 and P21–5 were located exclusively in a separated branch, which was distantly related to the other 5 known NSP4 genetic groups (Figure). However, a bootstrap support for the separation of the gene into a separate lineage is very strong with nucleotide sequencing but weak by amino acid analysis in this phylogentic tree. Our finding indicates that PoRV strains CMP034 and P21–5 are likely a novel NSP4 genetic group and, therefore, tentatively proposed as a NSP4 genetic group F.

Figure 1: Phylogenetic analyses of the NSP4 nucleotide (A) and amino acid (B) sequences displaying the relationships between porcine rotavirus strain CMP034 (shown in boldface), P21–5, and other 5 known NSP4 genetic groups. Bootstrap values are shown at the branch nodes. Branch length for a 10% nucleotide difference is indicated at the bottom.

Mentions:
The complete NSP4 nucleotide sequence of PoRV CMP034 strain was 750 bp and contained a single long open reading frame coding for a protein of 175 aa. Comparative analysis of the CMP034 NSP4 sequence with those of the 5 representative established genetic groups (A–E) showed the highest sequence identity, at 92.6% nt and 96.9% aa levels, with 1 PoRV strain, P21–5 (9). However, CMP034 and P21–5 shared a low degree of sequence identity with other NSP4 genetic groups. The NSP4 sequence identities of the CMPO34 and P21-5 strains ranged from 74% to 78% nt and 75%–79% aa levels with those of genetic group A; 77%–86% nt and 79%–86% aa levels with genetic group B; 69%–73% nt and 75%–78% aa levels with genetic group C; 62%–65% nt and 55%–60% aa levels with genetic group D; and only 43%–50% nt and 29%–33% aa levels with genetic group E. The phylogenetic tree confirmed that PoRV strains CMP034 and P21–5 were located exclusively in a separated branch, which was distantly related to the other 5 known NSP4 genetic groups (Figure). However, a bootstrap support for the separation of the gene into a separate lineage is very strong with nucleotide sequencing but weak by amino acid analysis in this phylogentic tree. Our finding indicates that PoRV strains CMP034 and P21–5 are likely a novel NSP4 genetic group and, therefore, tentatively proposed as a NSP4 genetic group F.

The PCR amplicon was sequenced in both directions by using the BigDye Terminator Cycle Sequencing kit (PerkinElmer-Applied Biosystems, Inc., Foster City, CA, USA) on an automated sequencer (ABI 3100; PerkinElmer-Applied Biosystems, Inc.)... The sequence of CMP034 was compared with those of reference strains available in the National Center for Biotechnology Information GenBank database by using BLAST (www.ncbi.nlm.nih.gov/blast)... The NSP4 nucleotide sequence of the CMP034 strain was deposited in GenBank under accession no... DQ534017... However, CMP034 and P21–5 shared a low degree of sequence identity with other NSP4 genetic groups... The phylogenetic tree confirmed that PoRV strains CMP034 and P21–5 were located exclusively in a separated branch, which was distantly related to the other 5 known NSP4 genetic groups (Figure)... The NSP4 sequence analysis of our CMP034 strain revealed a PoRV strain closely related genetically to the NSP4 gene sequence of PoRV strain P21–5 isolated in Slovenia... The only difference observed between the 2 strains was that CMP034 belonged to the G2-like genotype whereas P21–5 belonged to G1 genotype... The relatedness between NSP4 sequences of strains CMP034 and P21–5 was confirmed by phylogenetic analysis, which showed that both CMP034 and P21–5 clustered closely together in a branch separated from those of other 5 NSP4 genetic groups... This finding suggests that NSP4 of PoRV strain CMP034 and P21–5 may have derived from the same ancestor... The isolation of 2 strains of rotaviruses with a close genetic relatedness of NSP4 gene from Thailand and Slovenia, 2 countries that are located in different continents, may indicate that this novel NSP4 genetic group has already been introduced into PoRVs worldwide.